The present invention, in some embodiments thereof, relates to methods for ex-vivo and in-vivo culture of cells, and, more particularly, but not exclusively, to the use of devitalized, acellular micro-organ matrices for culture of cells, tissue engineering and therapeutic uses thereof.
Micro-Organs (MOs)
Micro-organs provide an in vivo-like culture system based on the preparation of small organ fragments whose geometry allows preservation of the natural epithelial/mesenchymal interactions and ensures appropriate diffusion of nutrients and gases to all cells. These organ fragments have been termed micro-organs (MOs) since they preserve the basic organ architecture but are of microscopic thickness (300 μm). The thickness of MOs assures that no cell is more than about 150-250 μm away from a source of nutrients and gases. Since the micro-architecture is preserved, the natural cell-cell and cell-ECM interactions are maintained. MOs derived from several organs can be cultured for long periods in a minimal medium, and can thrive in the absence of serum or exogenous factors. During such periods, MOs remain viable, maintain the basic organ microstructure and express tissue specific genes. Preparation and use of MO is further described in detail in U.S. Pat. No. 5,888,720, and PCT Applications No. IL03/00578, IL00/00365, IL00/00424, IL 01/00976 and US98/00594, all of which are incorporated herein by reference.
U.S. Pat. No. 7,297,540, incorporated herein by reference, by the present inventors concerns methods of generating tissue using devitalized, acellular scaffold matrices derived from micro-organs, using stem cells/progenitor cells of adult or embryonic origin. Such cell free, three-dimensional scaffolds have been termed Micro-organ derived matrices (MOMs).
MO-derived matrices (MOMs) preserve both the 3-D architecture and the molecular composition of the stroma of the organ of origin. Due to their specific microscopic thickness, MOMs ensure that no seeded cell will be more than 100-250 microns from a source of gases and nutrients.
Diabetes
Diabetes mellitus is one of the most common chronic diseases in the world. In the United States, diabetes affects approximately 16 million people—more than 12% of the adult population over 45. The number of new cases is increasing by about 150,000 per year. In addition to those with clinical diabetes, there are approximately 20 million people showing symptoms of abnormal glucose tolerance. These people are borderline diabetics, midway between those who are normal and those who are clearly diabetic. Many of them will develop diabetes in time and some estimates of the potential number of diabetics are as high as 36 million or 25-30% of the adult population over 45 years.
Diabetes and its complications have a major socioeconomic impact on modern society. Of the approximately $700 billion dollars spent on healthcare in the US today, roughly $100 billion is spent to treat diabetes and its complications. Since the incidence of diabetes is rising, the costs of diabetes care will occupy an ever-increasing fraction of total healthcare expenditures unless steps are taken promptly to meet the challenge. The medical, emotional and financial toll of diabetes is enormous, and increase as the numbers of those suffering from diabetes grows.
Type 1 diabetes is characterized by loss and dysfunction of beta cells. Although the success rate of islet-cell transplantation has increased with experience, poor engraftment and the paucity of available islets remain a major limitation to widespread use of transplantation. In addition, longitudinal analyses of islet recipients make it clear that transplanted islets fail to maintain function (e.g. glucose responsive insulin secretion) over time. Therefore, promotion of beta cell formation, in-vitro and in-vivo, is a major goal of type 1 diabetes therapy.
Although beta cell culture has been demonstrated, production of significant numbers of viable and functional (glucose responsive) cultured beta cells for transplantation has not been achieved. In various animal models, the importance of the microenvironment for replication and differentiation of beta cells and beta cell precursors has been demonstrated. For example, repeated passage of nestin-positive islet progenitors in-vitro, without GLP-1, results in loss of insulin secretion capability. Further, even insulinoma cells required supporting fibroblast culture and pituitary adenoma cell conditioned medium to maintain insulin secretion in long-term culture. Similarly, pancreatic endocrine progenitor cells failed to differentiate in-vitro without a synthetic matrix overlay and, despite in-vitro proliferation of the differentiated, insulin-secreting cells, they failed to engraft and form functional islets when transplanted in-vivo.
In highly evolved organisms epithelial cells are always supported by a connective tissue stroma. Epithelial-stromal interactions play an important role in the maintenance of the structure and function of epithelial cells, both during normal development and also in the adult organism. It has recently been shown that this particularly dense vascular network is required for proper endocrine function and islet size. It has also been shown that in vivo, islets are surrounded by a continuous peri-insular basement membrane that contains collagen IV and laminin, which is lost during islet purification. Indeed, adding growth factors and extracellular-matrix factors, including laminin, nicotinamide and insulin, can lead to the formation of ES-derived progeny in culture resembling cells committed to the pancreatic lineage.
Alternatives to the inefficient and unsuccessful culture of beta cells in static, two dimensional conditions have been suggested.
One of the most likely reasons for the lack of success thus far in islet cell culture and transplantation is that islet cells require efficient perfusion, and, after transplantation, islet tissue grafts are dependent on extensive neo-vascularization for survival.
Native islets in the pancreas have a rich vascular structure thought to provide efficient delivery of oxygen and nutrients to islet cells and ensure rapid dispersal of pancreatic hormones to the circulation [Jansson, L. & Carlsson, P. O. Diabetologia 45, 749-763 (2002); Menger, M. D., Yamauchi, J. & Vollmar, B. World J Surg 25, 509-515 (2001)], which is not preserved in culture. Further, intra-islet endothelial cells are lost following 7 days of islet culture [Mendola, J. F. et al. Transplant Proc 26, 689-691 (1994); Parr, E. L., Bowen, K. M. & Lafferty, K. J. Transplantation 30, 135-141 (1980)], thus cultured islets are susceptible to ischemic injury (e.g., lack of oxygen or nutrients) upon transplantation. Therefore, rapid and adequate islet revascularization may be crucial for the survival and function of transplanted islets [Zhang N et al., Am J Transplant 3, 1230-1241 (2003)].
U.S. Patent Application No. 20050048040 teaches a method for enhancing vascularization of islets by increasing the quantity and/or quality of endothelial cells residing within. U.S. Patent Application No. 20030113302 also teaches a method for enhancing vascularization of islets by contact thereof with endothelial cells in the presence or absence of a scaffold.
U.S. Patent Application No. 20060275900 to Presnell et al. envisions transdifferentiation of non-beta acinar cells to an insulin producing phenotype by culturing with pancreatic specific differentiation factors. Rotary culture systems have provided slightly greater expansion but are impractical. U.S. Patent Application 20090069903, to Shortkroff et al. discloses a double structured tissue implant, comprising a primary porous collagen scaffold with a secondary soluble collagen hydrogel scaffold surrounding the seeded islets. U.S. Patent Application No. 20090221068 to Kobayashi et al also proposes a biodegradable peptide hydrogel scaffolding for culture of hepatic and pancreatic cells, and its use for transplantation. U.S. Patent Application 20040126405 to Sahatjian et al. discloses the use of non-woven, synthetic polymers for transplanatation and/or organ reconstruction, for example, of islet cells. U.S. Pat. No. 7,427,415 and Application 20090004238 to Scharp et al. propose transplantation of beta cells and islets encapsulated within synthetic polymers. U.S. Patent Application No. 20080103606 to Berkland et al. discloses the implantation of multilayered groups of pancreatic islets affixed to a synthetic planar support. U.S. Patent Application No. 20090074732 to Badylak discloses the use of acid-decellularized, heat treated parenchymous tissue as a scaffold for tissue implantation. Badylak provides no reduction to practice, and does not teach micro-organ scaffolds of defined dimensions, maintaining the original tissue architecture. However, none of the proposed solutions succeeded in providing conditions for successful culturing of functional (e.g. glucose responsive insulin secretion) islets and/or beta cells suitable for long-term survival and function in-vivo.
It is estimated that less than 30% of transplanted islet mass becomes stably engrafted, despite the administration of a large quantity of islets per diabetic recipient [Boker A. et al., World J Surg 25, 481-486 (2001)]. Given the limited supply of cadaveric donors and the prevalence of type 1 diabetes, there is a widely recognized need for, and it would be highly advantageous to have, methods for both efficient ex-vivo culture of pancreatic islets, and for providing viable cultured islets suitable for long-term survival and function following transplantation.